1. Field of the Invention
The field of the invention is functional elements of human telomerasexe2x80x94an enzyme important in cell growth.
2. Background of the Invention
Telomeres are the dynamic nucleoprotein complexes that cap the ends of linear chromosomes. They prevent undesirable chromosome rearrangements and protect against genomic instability and the associated risk of carcinogenesis (Artandi and DePinho, 2000). Telomeres can also function as a mitotic clock that counts cell divisions by the gradual erosion of telomeric sequence. Telomere shortening forces cultured human primary cells to stop dividing when a critical minimum telomere length is reached (Colgin and Reddel, 1999). This entry into senescence acts as a protective checkpoint, guarding against genomic instability induced by telomere loss.
Many cellular factors are required to maintain telomere stability, including the telomere binding proteins that orchestrate a higher order telomeric chromatin structure (Collins, 2000). At a regulatory level, however, changes in telomere length appear to be accomplished primarily by activation or inhibition of telomerase. The telomerase ribonucleoprotein (RNP) extends chromosome 3xe2x80x2 ends by addition of one strand of tandem DNA repeats (Greider, 1995). Telomerases in all species share at least two components essential for catalytic activity: the telomerase reverse transcriptase protein (TERT) and the telomerase RNA (Bryan and Cech, 1999). Although TERTs share the reverse transcriptase (RT) active site motifs of viral RTs (Lingner et al., 1997), they are unique in their stable association with a telomerase RNA that contains the template for telomeric repeat synthesis Greider and Blackburn, 1989). Telomerase RNAs range in size from about 150-200 ucleotides (nt) in ciliates to greater than 1000 nt in yeasts.
In humans, misregulation of telomerase activity can have dire consequences. Telomerase activation accompanies tumorigenesis and is important for the continued viability of cultured human tumor cells (Kim et al., 1994; Artandi and DePinho, 2000; Collins, 2000). However, recent studies have suggested that telomerase activation in at least some human cell types is also essential for normal growth and development. Premature mortality caused by the X-linked disease dyskeratosis congenita (DKC) results from proliferative deficiencies and an increased risk of cancer in tissues which are normally highly regenerative, such as the skin and blood (Dokal, 1999). Cells from DKC patients have reduced levels of telomerase RNP and hastened telomere shortening (Mitchell et al., 1999b). This suggests that telomerase activation in highly proliferative tissues may be necessary to suppress potential genomic instability and to guarantee enough renewal capacity for a typical human lifespan. Together, these findings reveal that telomerase activation and inhibition must be carefully balanced to meet the proliferative demands of normal cells while at the same time guarding against the potential for unbridled proliferation of tumors. As a result, pharmacological methods for activating or restraining telomerase activity in vivo would both be useful.
The human telomerase RNA (hTR) is transcribed by RNA polymerase II and processed at its 3xe2x80x2 end to yield a mature transcript of 451 nt (Feng et al., 1995; Zaug et al., 1996; Mitchell et al., 1999a). The template for reverse transcription lies near the 5xe2x80x2 end of the molecule and specifies incorporation of the sequence TTAGGG to chromosome ends. In a previous study, we identified hTR primary sequence elements that are required for the stability and 3xe2x80x2 end processing of recombinant hTR in vivo (Mitchell et al., 1999a). Surprisingly, these elements form part of a structural motif shared with H/ACA small nucleolar (sno)RNAs, an RNA family that functions in the maturation of ribosomal RNA by directing cleavage and pseudouridine (xcexa8) formation (Tollervey and Kiss, 1997). Hybridization of a snoRNA to target RNA specifies the site of modification, while protein components of the stable snoRNP catalyze the reaction itself. Phylogenetic comparison of 35 vertebrate telomerase RNAs confirmed that the H/ACA motif is a universally conserved feature that is not present in ciliate or yeast telomerase RNAs (Chen et al., 2000). The 3xe2x80x2 half of hTR as bounded by the elements of the H/ACA motif (nt 211-451) can accumulate independently of the full-length molecule in vivo (Mitchell et al., 1999a). We refer to this region as the hTR H/ACA domain (see FIG. 1). When the hTR H/ACA domain is replaced with a heterologous H/ACA snoRNA, the chimeric RNA accumulates but does not support telomerase activity (Mitchell et al., 1999a). Therefore, sequences within the hTR H/ACA domain are critical for telomerase activity independent of the requirement for hTR stability in vivo.
Here, we define distinct motifs within the human telomerase RNA that contribute to telomerase RNP accumulation and activity. We find that hTR precursor processing, mature RNA accumulation, and H/ACA protein association are inseparably linked and require the consensus H/ACA motif elements within the H/ACA domain. Furthermore, we demonstrate that two regions within the telomerase RNA are required for telomerase activity in vivo and in vitro. One of these regions contains the template for reverse transcription as expected (hTR nt 1-209); the other is a telomerase-specific element within the H/ACA domain (hTR nt 241-330). Surprisingly, we find that both of these regions interact independently with TERT and bind to TERT in a largely noncooperative manner. Thus, a vertebrate-specific telomerase RNA motif physically separable from the template is required for telomerase activity. This work reveals an unexpected functional requirement for two distinct telomerase RNA-TERT interactions within the same telomerase RNP and establishes a fundamental difference between the structure of ciliate and vertebrate telomerase RNPs.
Relevant Literature
See U.S. Pat. Nos. 5,917,025 and 5,770,422.
Telomerase inhibition has utility as a clinical treatment for a broad range of human cancers (treated by telomerase inhibition) and age- or disease-induced cellular proliferative deficiencies (treated by telomerase activation). Requirements for telomerase function at a structural level have hitherto remained largely unknown. Here, we demonstrate the structural requirements for function of the essential human telomerase RNA component (hTR) in vivo and in vitro. Two types of function for RNA elements are discriminated. First, we have identified RNA elements that are essential for RNA stability in vivo but are dispensable for catalytic activity in vitro. Second, we have identified RNA motifs that are critical for catalytic activity in vivo and in vitro.
The first category, RNA elements essential for RNA stability in vivo, includes all elements of the consensus H/ACA motif in proper sequence context (5xe2x80x2 terminal stem: nts 211-214 paired to 367-370; H box: unpaired nts 372-377; 3xe2x80x2 terminal stem: nts 381-384 paired to 440-443; ACA box: unpaired nts 446-448) and one additional element (3xe2x80x2 stem-loop: nts 411-418). Cellular accumulation of stable hTR, dependent on the H/ACA motif elements described above, is coincident with the association of RNA with H/ACA proteins (as assayed by association with the protein dyskerin, as a cooperative assembly of the proteins dyskerin, hNhp2, hNop10 and additional cooperative or noncooperative assembly of the protein hGAR1.
The second category, RNA elements essential for catalytic activity both in vivo and in vitro includes a region containing the template (nts 1-208 in vivo and in vitro; in vitro element minimized to nts 44-186) and a second region termed IH1 (nts 241-330 in vivo and in vitro; in vitro element minimized to 253-322 without 271-285). Each of these RNA elements binds to the telomerase reverse transcriptase protein (TERT) independently, both in vivo and in vitro. However, binding of both elements is required for catalytic activity, in vivo and in vitro.
The first 656 amino acids of hTERT are sufficient for binding to both RNA elements required for catalytic activity with efficiency similar to full-length hTERT in vivo. In vivo methods for stable recombinant RNA production and recombinant protein-RNA interaction assays, modifiable to provide in vivo reporter assays for RNA structure and/or RNA-protein interaction and described below and in Cheng et al. (1999) Mol Cell Biol 19, 567-576.
In vitro methods for analysis of requirements for recombinant RNA activity and recombinant RNA-protein interaction, optimization of telomerase catalytic activity by RNA minimization, trans complementation, and titration; and production of reconstituted telomerase RNPs limited in activity by a specific RNA binding requirement are described below.
This work enables screening technology to develop compounds with clinical utility for telomerase inhibition or activation. First, important RNA structures have been determined. Thus, screens for molecules that interact with an essential hTR sequence and/or structure are used to identify candidate modulators of telomerase stability and/or activity. Second, protein interactions mediated by essential hTR elements have been determined. Thus, screens for molecules that affect protein-RNA interaction are also used to identify candidate modulators of telomerase stability and/or activity.
Improvements/distinctions from current screening technology using nucleotide incorporation activity assays:
1. Catalytic activity in vivo on telomeres and in vitro on oligonucleotide substrates have been demonstrated to have different requirements for substrates and enzyme components; therefore, molecules that inhibit activity in vitro may not do so similarly in vivo. In addition, the active site of an endogenous telomerase holoenzyme RNP may differ from that of partially purified holoenzyme or recombinant core enzyme isolated or produced in vitro. In contrast, with the disclosed functional domains, RNA or protein-RNA interaction based screens can use RNA elements demonstrated to be required in vivo and furthermore known to be required in a particular structural context.
2. Because at least some features of the telomerase active site are shared by polymerases in general, a large number of activity inhibitors will have multiple targets in vivo. In contrast, the disclosed telomerase RNA motifs are either entirely unique (the ones required for activity) or at least partially distinct in sequence (the ones required for stability) from all other known human RNAs.
3. Molecules attained by screening for activity inhibition and by screening for RNA structure or protein-RNA interaction impact both have potential to reduce the level of telomerase enzyme or enzyme activity. However, binding of an activity inhibitor is more likely to be reversible, whereas disruption of a RNA structure or protein-RNA interaction may also lead to mislocalization or turnover and thus more permanent inactivation.
4. The new technology can be used to improve catalytic activity-based screens, by allowing smaller RNAs to be used at defined concentrations in sensitized activity assays with recombinant telomerase protein(s).
Accordingly, the invention provides methods and compositions relating to discrete elements of human telomerase and human telomerase RNA. In one embodiment, the invention provides a polynucleotide comprising only one element of human telomerase RNA, wherein the element consists of SEQ ID NO:1, residues 241-330. Such human telomerase RNA elements may be employed in mixtures with a human telomerase polypeptide such as TERT or dyskerin, wherein the polypeptide and polynucleotide specifically interact, and such mixtures may be employed in methods for identifying modulators of a human telomerase polypeptide - human telomerase RNA interaction. In a particular embodiment, such methods comprise the steps of incubating the mixture with a candidate agent under conditions whereby, but for the presence of said agent, said polypeptide specifically binds said polynucleotide at a reference affinity; and detecting the binding affinity of the polypeptide to the polynucleotide to determine an agent-biased affinity, wherein a difference between the agent-biased affinity and the reference affinity indicates that the agent modulates the binding of the polypeptide to the polynucleotide. The binding affinity may be detected directly by solid phase binding assay or inferentially as telomerase catalytic activity, and the mixture may be in vitro or within a viable cell.